Gastric inhibitory peptide (GIP) was first isolated from porcine small intestine and was originally named for its ability to inhibit acid secretion. Subsequent analysis of its physiological properties has demonstrated that GIP is a potent stimulator of endogenous insulin release and that it plays an important role in maintaining glucose and lipid homeostasis. Recent studies in patients with type II diabetes mellitus have demonstrated that although nutrient-stimulated GIP release appears to be normal, the insulinotropic effect of exogenous GIP is greatly diminished, suggesting that GIP may not be functionally active. Most gastrointestinal regulatory peptides undergo posttranslational processing prior to becoming biologically active. Although GIP was first recognized as a 42 amino acid polypeptide, recent studies performed in this laboratory indicated that a smaller alpha-amidated form of GIP(1-30) possesses similar insulinotropic effects to GIP(1-42). Furthermore, a glycine-extended prohormone followed by a dibasic Lys-Lys residue has been identified in the GIP cDNA sequence. It is thus likely that proGIP undergoes posttranslational C-terminal alpha-amidation modification before becoming biologically active. Moreover, it is also possible that abnormal posttranslational processing of proGIP could play a role in the pathogenesis of type II diabetes mellitus. To date, however, no information regarding posttranslational processing of the C-terminus of GIP is available. To determine the importance of posttranslational processing of proGIP, this project will: (1) define the biologically active portions of proGIP by examining the effects of different peptide fragments of proGIP in stimulating insulin release from a rat insulinoma cell line (RIN-5AH); (2) examine the postranslational processing of the C-terminus of proGIP in the rat duodenum and salivary glands, organs known to express the GIP gene, during both basal and stimulated (glucose fed) states, using region- specific antibodies against different peptide fragments of proGIP; (3) determine the molecular mechanisms regulating posttranslational processing of the C-terminus of proGP by transfecting endocrine and small intestinal cell lines with wild type and mutant (by site-directed mutagenesis) GIP DNA. Collectively, the information gained from this proposal will add a significant new dimension to our understanding of the molecular mechanisms involved in posttranslational processing of proGIP. Moreover, because of the close structural and functional relationship of GIP to other GI peptides, such studies will advance our general understanding of the molecular mechanisms involved in nutrient-related hormone processing. Finally, these studies should enhance our understanding of the biology of GIP and of the possibility of GIP contributing to the pathogenesis of diseases characterized by abnormalities in gastric acid secretion and glucose homeostasis.